JPS63235301A - Polysaccharide having heparinoid activity, its preparation, and anticoagulant containing the same - Google Patents

Abstract

PURPOSE:To prepare the title polysaccharide which has a strong heparinoid activity and small side effects, by extracting a green alga belonging to the genus Monostroma with water or hot water. CONSTITUTION:A dried alga belonging to the genus Monostroma (e.g. Monostroma) is dipped in about 10 times the amount of water, and the swollen alga is cut into fine pieces and then freed of water and the filtrate. The resulting alga is extracted with 5-10 times by weight of hot water at 60-150 deg.C for 10min-5hr, after which the water, filtrate and hot water are dialyzed, and the resulting product is concentrated at a low temperature of freeze-dried to give a polysaccharide which has a heparinoid activity and has the chemical and physical properties shown in the attached Table.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a polysaccharide having heparinoid activity obtained from green algae of the genus Humane, a method for producing the same, and an anticoagulant containing the same.

Here, the heparinoid activity refers to the anticoagulation effect on blood shown by antithrombin activity, similar to that of heparin.

(Prior Art) Conventionally known polysaccharides having heparinoid activity include heparin, dextran sulfate sodium salt (hereinafter abbreviated as dextran sulfate), fucoidan, and the like.

Although heparin is widely distributed in various tissues of higher animals, it is present in large quantities in the liver and mast cells, and is obtained by extraction from these cells, and is usually handled as a sodium salt. Its structure consists of D-glucosamine residue and hexuronic acid (D
-glucuronic acid and L-iduronic acid) residues alternately 1,4
It has a bonded skeleton.

Biologically, it has the effect of inhibiting blood coagulation and is used to prevent blood coagulation or thrombosis. Furthermore, since it has the action of clarifying plasma lipids by activating riboprotein lipase, it is sometimes used to treat hypercholesterinemia and arteriosclerosis.

Dextran sulfate is a sulfate ester of α-1,6-glucan, and was synthesized in 1945 by Gr6wall et al. Among them, those with a molecular weight of about 6,500 and a sulfur content of 16 to 18% are used clinically because they have anticoagulant and plasma clarifying effects similar to heparin, and are low in toxicity.

Furthermore, fucoidan is a sulfated polysaccharide of brown algae whose main component is fucose, and although much of its structure is unknown, it is known to have an anticoagulant effect similar to heparin. However, since fucoidan is not manufactured and used industrially, it has not been put to practical use as a blood coagulation inhibitor.

(Problems to be Solved by the Invention) However, these known polysaccharides having heparinoid activity have had various problems as shown below.

Heparin is manufactured by extracting and purifying the internal organs of higher animals, but there have been problems with its quality and manufacturing method.

In other words, because animal tissue is used as a raw material, unpleasant odors are generated during production, a lot of cost is required to process the extraction residue, and contaminants with similar molecular weights (β-heparin, etc.) are mixed in the extract. Therefore, there were manufacturing problems such as the need for many steps to obtain a product with a specific molecular weight.

Furthermore, it is extremely difficult to keep the anticoagulant activity of the product at a certain level, and it is necessary to compare the activity with standard heparin for each loft, and if a strong anticoagulant effect is required, It also had problems in use, such as the need to use large amounts.

On the other hand, after the discovery of heparin, methods for synthetically producing polysaccharides with anticoagulant effects were investigated.

As a result, dextran sulfate was produced by synthesis, but this also had many problems.

In other words, during the synthesis of dextran sulfate, it is necessary to collect both components of the appropriate molecular weight after the esterification reaction, and furthermore, the amount of sulfuric acid must be adjusted to an appropriate amount, which is a manufacturing difficulty. , ■ The heparinoid activity of dextran sulfate is often relatively weak compared to that of heparin, and it generally needs to be used in large amounts; If the amount of the compound is large, there are problems such as side effects such as irritation may occur after intravenous injection.

In addition to these, many sulfated polysaccharides have been developed and investigated as anti-coagulants, but all have been found to have anti-coagulant activity comparable to or inferior to that of heparin.

Based on the above, there has been a strong desire for a heparinoid-active polysaccharide that has stronger heparinoid activity, fewer side effects, and fewer problems in production.

(Means for Solving the Problems) As a result of many years of research on seaweed polysaccharides, the present inventors discovered that polysaccharides containing rhamnan sulfate as a main component obtained from green algae of the genus Aminosa have high heparinoid activity. ,
The present invention was completed by confirming the molecular weight, sulfate group content, and factors that influence heparinoid activity of polysaccharides having heparinoid activity.

That is, the present invention provides a novel polysaccharide having heparinoid activity (hereinafter sometimes referred to as the polysaccharide of the present invention) obtained from, for example, green algae of the genus Atomina, a method for producing the same, and an anticoagulant containing the same. It is something to do.

The polysaccharide of the present invention is produced by extracting a green alga of the genus Aminosa with water or hot water, collecting a polysaccharide having heparinoid activity from the extract, and purifying the polysaccharide.

Examples of the green alga of the genus Monostroma that can be used as the raw material of the present invention include Monostroma niti
dumWittrock), Ezohitoegusa (M, an
gicava Kjellman), Hirohanohitoegusa (M, latissium Wittrock), Motsukihitoe ('M, zostericola Ti1d)
Examples include green algae belonging to the genus Humanegusa such as E. en), but Homophylla or Hirohanohenogusa is preferred from the viewpoint of economic efficiency and heparinoid activity, and among these, polysaccharides obtained from Homoecium are particularly preferred because they exhibit the highest heparinoid activity.

To obtain an extract from green algae of the genus Aminosa, it can be carried out, for example, as follows.

The raw material, green algae of the genus Hitodensis, is generally transported and stored as a dry product because the algae bodies are soft and easily rot. Therefore, when extracting a polysaccharide having heparinoid activity from the green algae, it is easier to extract the algae by soaking the algae in water to swell it in advance. In order to swell the algae, for example, if the algae has been dried to a moisture content of 5%, it is desirable to immerse the algae in distilled water in an amount approximately 10 times the weight of the algae.

Thereafter, the algae are shredded and water-extracted algae are obtained by filtration or centrifugation. At this time, a small amount of anti-blood coagulation polysaccharide is present in the water in which the algae were soaked and in the filtrate after shredding. but,
Its heparinoid activity is weak, and when the activity of heparin is 1, its activity is about 0.5 to 1. Moreover, the amount is 0.5 to 2 when the weight of algae is 100.

The shredding method can be any method that normally shreds seaweed, but since algae is soft, a device such as a juicer, mixer, or homogenizer can be used.

Next, heparinoid active polysaccharide is extracted from the shredded and water-extracted algae with hot water. The amount of hot water is 5 to 10 times the amount of algae.
The magnification, temperature and time are respectively 60℃ or higher and 150℃ or lower,
The time is preferably 10 minutes or more and 5 hours or less.

The activity of the heparinoid active polysaccharide in the extract thus obtained is 1.05 to 4 when the activity of heparin is 1.
That's about it. Moreover, the amount is 6 to 10 when the weight of algae is 100. This polysaccharide has stronger activity than conventional sulfate polysaccharide having heparinoid activity and can be used as is.

Furthermore, by collecting and purifying a polysaccharide having heparinoid activity from the extract thus obtained, a highly active polysaccharide can be obtained.

For collection and purification, for example, the water or hot water extract obtained above is subjected to dialysis, followed by low-temperature concentration or freeze-drying, preferably DE! It is retained in a column such as AE ion-exchange cellulose column chromatography, eluted with an eluent, separated into various fractions, and dried by a method such as freeze-drying to obtain a highly active anti-blood coagulation polysaccharide.

Suitable eluents are, for example, water and 0.5 to 2.5 molar potassium chloride solutions. As for the elution method, both methods of continuously changing the concentration of potassium chloride in the eluent and methods of changing it stepwise are possible, but
In order to accurately separate fractions, a method in which the concentration of potassium chloride is changed stepwise is more preferable. In addition, it is desirable to limit the number of times of separation using the column to one time if you want to increase the yield, but if you want high activity, the same operation can be repeated two or more times. . When the number of times of column separation is 1, the heparinoid activity is about 2.5 to 4, and when the number of column separation is 2 times, the heparinoid activity is about 3 to 8.

The polysaccharide of the present invention obtained as described above has the following physical and chemical properties.

B) Color and Shape: The color changes depending on the degree of purification of the polysaccharide of the present invention, but in general, it tends to be pale yellow to yellowish brown when the degree of purification is low, and white when the degree of purification is high. The shape may change from powder to spiral depending on the degree of dryness, and if the moisture content is low, it becomes powder. It is preferable to store it in a dry powder form because if too much moisture is left behind during storage, mold may develop.

) Molecular weight: Molecular weight is determined by gel filtration column chromatography (e.g. Toyobar HW-65
) or when determined by light scattering measurement, it varies depending on the degree of purification of the polysaccharide of the present invention. If the degree of refining is low, it will be 50,000 to 300,000, and if the degree of refining is high, it will be 80,000 to 30,000.
It becomes 00,000.

Sulfate ester content: The polysaccharide of the present invention contains a sulfate ester as part of its structure, which is a functional group indispensable for the expression of anticoagulant activity. The content of sulfate ester is suitably 5 to 35% based on the dry weight of the polysaccharide of the present invention for the expression of anticoagulant activity, and more preferably 7 to 30%.

2) Sugar composition: The sugar composition of the polysaccharide of the present invention is mainly composed of rhamnose and glucuronic acid, and the composition ratio thereof is most preferably in the range of rhamnose:glucuronic acid = 1:1 to 9:1.

The proportion of glucuronic acid in -S tends to decrease as the degree of purification of the polysaccharide of the present invention increases. Furthermore, there are other substances contained as wis such as arabinose, xylose, mannose, glucose, and galactose, but their amounts are relatively small and the amount of each is about O-0.3 when rhamnose is 1. .

N) Solubility: Easily soluble in water, easily soluble in dimethyl sulfoxide, soluble in 0-6N hydrochloric acid, sulfuric acid or nitric acid, soluble in 0-20% ethyl alcohol, soluble in 0-3N sodium hydroxide, Insoluble in benzene and cyclohexane) Protein content: The protein content per dry weight of polysaccharide is 0 to 25%, but it varies depending on the degree of purification of the polysaccharide, and the protein content decreases as the degree of purification increases. .

g) The infrared absorption spectrum is as shown in FIG.

H) Heparinoid activity: Specific activity is 1 when heparin's anticoagulant activity is 1.
．． 05-8.

b) Specific optical rotation: The specific optical rotation is [α]25D=-35''' to -75°.

When preparing an anti-blood coagulation preparation using the polysaccharide of the present invention, it is only necessary to contain the product of the present invention, and the condition and shape of the preparation, as well as the mixing with other components, can be adjusted as desired. It is possible. For example, similar to heparin preparations, it can be prepared in the form of powder, liquid, ointment, etc., and can be administered to patients before surgery, mixed with various blood products, or used in blood collection tubes. It can also be applied to.

In any case, since the heparinoid activity is relatively high, it is possible to exert anticoagulant activity with a smaller amount than conventional sulfated polysaccharides.

(Example) Next, the present invention will be explained with reference to Examples.

Example 1 1) Monostroma nitid
ulII Witrock) 100g (moisture 5.0%
) in 1 liter of distilled water for 30 minutes to swell.

2) Place the algae and water obtained in 1) in a commercially available mixer for approximately 5 minutes.
The algae were shredded and left to stand for 30 minutes, and then filtered through three layers of gauze to separate algae bodies-1 and liquid-1 (solid content: 1.2 g).

3) Add 700 cc of hot water to the algae body-1 obtained in 2), heat it at 70-100°C for 2 hours, cool it to room temperature, and separate the algae body-2 and filtrate-2 ( Solid content 8.2g
) and separated.

4) The filtrate-2 obtained in 3) was centrifuged at 400 rpm for 20 minutes to remove a slight amount of residue, and after 3 days of dialysis against running water using a cellophane membrane, the mixture was heated to 5 tmH.
It was freeze-dried under conditions of 1 day to obtain crude heparinoid active polysaccharide (PolyW-1).

5) 150 mj of the heparinoid active multi-F-1 (Ig) obtained in 4)! of water and centrifuged the water-insoluble residue (40
00 rpm for 20 minutes) and the supernatant was washed with Dt! It was injected into a column (diameter 4.5 cs x length 55 ca+) packed with AE-cellulose (OH type).

Next, about 1.5 liters of distilled water was passed through the column to flush out the non-adsorbed polysaccharide fraction. The end point of elution with water was defined as the point at which no phenol-sulfuric acid reaction was observed (fraction-1).

Next, 0.5 mol, 0.7 mol, and 2.0 mol potassium chloride aqueous solutions were added to the column at 10100O! The C1 fraction was run until no phenol-sulfuric acid reaction was observed, and both fractions were collected (fraction -2, 3, and 42°. Both fractions were dialyzed against running water for 3 days using a cellophane membrane, and then freeze-dried. And polysaccharide-
1', 2', 3', 4' each at 45% relative to polysaccharide-1,
Amounts of 27%, 2.7% and 3.0% were obtained.

6) Dissolve the polysaccharide-2'600■ obtained in 5) in 10011 Il of water and inject it again into a DEAE-ion exchange cellulose column (diameter 4.5 cm, length 55 aa), and adjust the concentration of potassium chloride from 0 to 0. It was varied linearly up to .27 mol and eluted at a flow rate of 1.2 mJ/min.

The eluate was divided into two portions of 20 Ill, and the color was developed by the phenol-sulfuric acid method, and the absorbance at 480 n+s was measured to obtain the chart shown in FIG.

Both portions were combined into A, B, C, and D as shown in Fig. 1, and each was dialyzed and freeze-dried to obtain PolyW-5, 6, 7, and 8.

Test Example 1 The anticoagulant activity of each sample obtained in Example 1 was investigated by the following method, and the results shown in Table 1 below were obtained.

Method for measuring blood coagulation inhibitory activity: Blood coagulant inhibitory activity was determined as antithrombin activity in an in vitro O system containing heparin cofactor (see Toho Medical Journal, Vol. 20, p. 939, 1971).

Thrombin in 1 ml of 1% saline solution of bovine serum as heparin cofactor and 1 ml of saline solution of thrombin
was added and mixed with 1ml of 20mM barbital buffer, and then immersed in a 37°C water bath for 30 minutes to reach temperature equilibrium. This mixed liquid is 0.3mj! To this, 0.1 mβ of 1% physiological saline containing fibrinogen was added, and the time (71 seconds) from immediately after this to the formation of fibrin was determined and used as a blank test.

In this system, solutions 2II11 of 20mM barbital buffer containing seaweed polysaccharides with different concentrations were added, and after temperature equilibration at 37°C, the time until fibrin formation (72 seconds) was determined by the same procedure as described above. Since this takes a longer time than the previous TI seconds due to thrombin activity being inhibited, the activity was indicated by the time difference between the two (T2-T1 seconds). These measurements were performed at each sample concentration, and the time to fibrin formation was
The average value was determined from five or more measurements.

As a control experiment, the activity was measured using a heparin barbital buffer solution (3 ml/100 ml!) under similar conditions, and the activity was expressed as a relative value to this activity.

Note that ND in the table means not detected.

(The following is a blank space) The following points are recognized from the infrared absorption spectrum (Fig. 2) of Multi#a-6.

(2) Since absorption corresponding to S-0 at 1240 nm and C-0-S at 855 nm is observed, sulfate ester exists in polysaccharide-6.

Moreover, this sulfate ester is determined to have an axial orientation based on the infrared absorption of C-0-S stretching at 855 nm.

■ Heparin has absorptions corresponding to N-H groups at 1440 nm and 1550 nm, whereas multi-I! -Not in 6.

From this fact, it is determined that polyW-6 does not contain amino sugars as constituent sugars and that the type of sulfate ester present is not a sulfamino bond. This further suggests that Elson-M
This is also supported by the results of the organ reaction.

Test Example 2 The solubility of polysaccharide-6 in various solvents was investigated by the following method.

Multi-1i-6100■ solvents shown in Table 2 1 mA! After stirring at room temperature for 5 minutes, the presence or absence of a precipitate was observed. The results are also shown in Table 2.

In addition, in the table, ○ indicates soluble, Δ indicates slightly soluble, and × indicates insoluble.

Table 2 (Effects of the Invention) As described above, the polysaccharide having heparinoid activity of the present invention has a stronger anti-blood coagulation effect than the conventional sulfated polysaccharide for anti-blood coagulation, and exhibits a blood coagulation effect with a relatively small amount. Since it is manufactured from inexpensive raw materials, it is highly economical.

Furthermore, the production method of the present invention has a weaker odor than conventional heparin raw materials and does not generate any unpleasant odor.

Furthermore, it is only necessary to extract and purify the polysaccharide, and there is no need to adjust the sulfuric acid content as in the dextran sulfuric acid manufacturing process. It is possible to overcome many manufacturing problems such as

[Brief explanation of the drawing]

Figure 1 shows multi-F-2' ion exchange chromatography.
It is a drawing showing a gradient elution curve. Figure 2 is many! It is a drawing showing the infrared absorption spectra of -6 and heparin.

Claims (1)

[Scope of Claims] 1. A polysaccharide having heparinoid activity and having the following physical and chemical properties obtained from green algae of the genus Homoecium. b) Color and shape: white to yellowish brown powder b) Solubility: Easily soluble in water, easily soluble in dimethyl sulfoxide,
Soluble in 0 to 6N hydrochloric acid, sulfuric acid or nitric acid, Soluble in 0 to 20% ethyl alcohol, Soluble in 0 to 3N sodium hydroxide, Insoluble in benzene and cyclohexane C) Sugar composition: Mainly rhamnose and glucuronic acid. Ingredients and composition ratio: Rhamnose: Glucuronic acid = 1:1 to 9
:1 d) Molecular weight cutoff: 50,000 to 5,000,000 It gives a single to three peaks in gel filtration column chromatography and has a molecular weight of 50,000 to 300,000 by the chromatography and light scattering method. e) Sulfate ester content: The sulfate ester content is 5 to 35% based on the dry weight of the polysaccharide. f) Protein content: The protein content is 0 to 25% per dry weight of polysaccharide. g) The infrared absorption spectrum is as shown in FIG. h) Heparinoid activity: increase the anti-coagulant activity of heparin by 1
The specific activity is 1.05 to 8. li) Specific optical rotation: The specific optical rotation is [α]^2^3_D=-35
°~-75°. 2. A method for producing a polysaccharide having heparinoid activity, which comprises collecting a polysaccharide having heparinoid activity having the following physicochemical properties from an extract extracted from green algae of the genus Aminosa with water or hot water. b) Color and shape: white to yellowish brown powder b) Solubility: Easily soluble in water, easily soluble in dimethyl sulfoxide,
Soluble in 0 to 6N hydrochloric acid, sulfuric acid or nitric acid, Soluble in 0 to 20% ethyl alcohol, Soluble in 0 to 3N sodium hydroxide, Insoluble in benzene and cyclohexane C) Sugar composition: Mainly rhamnose and glucuronic acid. Ingredients and composition ratio: Rhamnose: Glucuronic acid = 1:1 to 9
:1 d) Molecular weight cutoff: 50,000 to 5,000,000 It gives a single to three peaks in gel filtration column chromatography and has a molecular weight of 50,000 to 300,000 by the chromatography and light scattering method. e) Sulfate ester content: The sulfate ester content is 5 to 35% based on the dry weight of the polysaccharide. f) Protein content: The protein content is 0 to 25% per dry weight of polysaccharide. g) The infrared absorption spectrum is as shown in FIG. h) Heparinoid activity: increase the anti-coagulant activity of heparin by 1
The specific activity is 1.05 to 8. li) Specific optical rotation: The specific optical rotation is [α]^2^5_D=-35
°~-75°. 3. An anti-blood coagulant characterized by containing as an active ingredient a polysaccharide having heparinoid activity and having the following physicochemical properties obtained from green algae of the genus Homoecium. b) Color and shape: white to yellowish brown powder b) Solubility: Easily soluble in water, easily soluble in dimethyl sulfoxide,
Soluble in 0 to 6N hydrochloric acid, sulfuric acid or nitric acid, Soluble in 0 to 20% ethyl alcohol, Soluble in 0 to 3N sodium hydroxide, Insoluble in benzene and cyclohexane C) Sugar composition: Mainly rhamnose and glucuronic acid. Ingredients and composition ratio: Rhamnose: Glucuronic acid = 1:1 to 9
:1 d) Molecular weight cutoff: 50,000 to 5,000,000 It gives a single to three peaks in gel filtration column chromatography and has a molecular weight of 50,000 to 300,000 by the chromatography and light scattering method. e) Sulfate ester content: The sulfate ester content is 5 to 35% based on the dry weight of the polysaccharide. f) Protein content: The protein content is 0 to 25% per dry weight of polysaccharide. g) The infrared absorption spectrum is as shown in FIG. h) Heparinoid activity: increase the anti-coagulant activity of heparin by 1
The specific activity is 1.05 to 8. li) Specific optical rotation: The specific optical rotation is [α]^2^5_D=-35
°~-75°. 4. The anti-blood coagulant according to claim 3, wherein the dosage form is one selected from the group consisting of an intravenous injection solution, an intramuscular injection solution, an ointment, and a powder.